Device and method for purifying sample containing DNA

Abstract

The invention relates to a device and a method for purifying a DNA-containing sample, the device comprises: a sample chamber used for containing a DNA-containing sample to be treated, a first semipermeable membrane arranged at a first end and a second semipermeable membrane arranged at a second end, the first semipermeable membrane and the second semipermeable membrane being in membrane structures, and the first semipermeable membrane and the second semipermeable membrane being in a direction from the inner side to the outer side of the sample chamber, the first end and the second end can pass through a non-target isolate but cannot pass through a target isolate, an electrophoretic field is formed between the first end and the second end, the target isolate is DNA, and the non-target isolate comprises charged impurities; the first ends of the first liquid storage device and the second liquid storage device are sealed in the first liquid storage device, the second ends of the first liquid storage device and the second liquid storage device are sealed in the second liquid storage device, and the first liquid storage device and the second liquid storage device are used for containing an electrophoretic buffer solution; an electrode on one side of the electrode assembly extends into the first liquid storage device, an electrode on the other side of the electrode assembly extends into the second liquid storage device, a loop is formed, and a sample containing DNA is subjected to electrophoresis. By means of electrophoresis, impurities and DNA are migrated together, the DNA is intercepted through the semi-permeable membrane, the impurities can be removed through the semi-permeable membrane, and the purposes of removing the impurities such as protein and polysaccharide and purifying the DNA are achieved. Due to the fact that a mild impurity removal mode is adopted, operation such as violent vibration is avoided, the possibility that DNA breaks in the purification process is reduced, and the purposes of purifying DNA and keeping the length of the DNA at the same time are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a device and method for purifying a sample containing DNA. Background Art

[0002] DNA purification is a key step in many molecular biology and biochemistry techniques, used to isolate and purify DNA molecules from biological samples. The DNA purification process can remove other biological molecules such as proteins, RNA, polysaccharides, etc., thereby obtaining high-quality DNA samples for subsequent experiments and analysis.

[0003] At present, there are many DNA purification technologies, such as phenol / chloroform method, silica gel column purification method, magnetic bead purification method, salting out method, etc., but they all have their own disadvantages. The phenol / chloroform method is cumbersome to operate and requires contact with toxic reagents, which is harmful to the health of experimenters. The silica gel column purification method is prone to DNA breakage when purifying ultra-long fragment DNA, and the DNA bound to the silica gel column is not easy to elute, and the recovery rate is low. The magnetic bead purification method is also prone to DNA breakage when purifying ultra-long fragment DNA, and the magnetic bead DNA is prone to aggregation and difficult to elute. When purifying DNA by the salting out method, impurities such as proteins and polysaccharides are easily precipitated together with the DNA, resulting in a low purity of the obtained DNA, which is not suitable for applications with high requirements for DNA purity.

[0004] Therefore, how to provide a device and method for purifying a sample containing DNA to obtain long fragments of DNA with good purity is a technical problem that needs to be solved urgently in this field.

[0005] Glossary:

[0006] Forward and reverse electrophoresis: electrophoresis in which the voltage remains unchanged but the voltage direction alternates between forward and reverse over time.

[0007] Unidirectional electrophoresis: electrophoresis in which the voltage direction remains unchanged. Summary of the invention

[0008] In view of this, the present invention provides a device for purifying a sample containing DNA to obtain long fragments of DNA with good purity. In addition, the present invention also provides a method for purifying a sample containing DNA using the above device.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A device for purifying a sample containing DNA, comprising:

[0011] A sample chamber, wherein the sample chamber is used to hold a sample containing DNA to be processed, a first semipermeable membrane is provided at a first end of the sample chamber, a second semipermeable membrane is provided at a second end of the sample chamber, and both the first semipermeable membrane and the second semipermeable membrane are membrane structures, and non-target separated substances can pass through the direction from the inside to the outside of the sample chamber, but target separated substances cannot pass through, an electrophoresis field is formed between the first end and the second end of the sample chamber, the target separated substance is DNA, and the non-target separated substances include charged impurities;

[0012] A first liquid reservoir and a second liquid reservoir, wherein the first end of the sample chamber is sealed in the first liquid reservoir, the second end of the sample chamber is sealed in the second liquid reservoir, and the first liquid reservoir and the second liquid reservoir are used to contain electrophoresis buffer solution;

[0013] An electrode assembly, wherein one electrode of the electrode assembly extends into the first liquid reservoir, and the other electrode of the electrode assembly extends into the second liquid reservoir to form a loop for electrophoresis of a sample containing DNA.

[0014] Preferably, in the above-mentioned device for purifying a sample containing DNA, the electric field strength between the two electrodes of the electrode assembly is 3V / cm-14V / cm, and the electrophoresis time maintained by the electrode assembly is 30min-60min.

[0015] Preferably, in the above-mentioned device for purifying a sample containing DNA, the electric field strength between the two electrodes of the electrode assembly is 7 V / cm, and the electrophoresis time maintained by the electrode assembly is 60 min.

[0016] Preferably, in the above-mentioned device for purifying a sample containing DNA, the electrode assembly is an electrode assembly that can alternately generate a forward current and a reverse current.

[0017] Preferably, in the above-mentioned device for purifying a sample containing DNA, when the electrophoresis condition is forward and reverse electrophoresis, the time length for which the electrode assembly maintains forward electrophoresis is longer than the time length for which the electrode assembly maintains reverse electrophoresis.

[0018] A method for purifying a sample containing DNA, using any of the above-mentioned devices, comprising:

[0019] placing a sample containing DNA in the sample chamber, and adding electrophoresis buffer to the first reservoir and the second reservoir;

[0020] Applying an electric field between two electrodes of the electrode assembly to perform forward and reverse electrophoresis;

[0021] The liquid in the sample chamber after electrophoresis is further purified to obtain DNA.

[0022] Preferably, in the above method for purifying a sample containing DNA, the duration of the forward electrophoresis of the forward and reverse electrophoresis is longer than the duration of the reverse electrophoresis.

[0023] Preferably, in the above method for purifying a sample containing DNA, the forward electrophoresis and the reverse electrophoresis of the forward and reverse electrophoresis are performed alternately at least twice, and within a single cycle, the total duration of the forward electrophoresis is greater than the total duration of the reverse electrophoresis.

[0024] Preferably, in the above method for purifying a sample containing DNA, the step of further purifying the liquid in the sample chamber after electrophoresis comprises: using ethanol or an ethanol aqueous solution to precipitate the DNA, the volume ratio of the ethanol or ethanol aqueous solution to the liquid in the sample chamber being (1-3): 1, and the volume fraction of ethanol in the ethanol aqueous solution being not less than 90%;

[0025] And / or, the step of further purifying the liquid in the sample chamber after electrophoresis further includes: washing the DNA precipitate with an ethanol aqueous solution, the number of washings is 1 to 3 times, and the volume fraction of ethanol in the ethanol aqueous solution is not less than 75%;

[0026] And / or, the cleaning further includes a dissolving step, wherein the dissolving solution is water, EB solution, Tris-HCl solution or TE buffer;

[0027] And / or, the electrophoresis buffer uses at least one of Tris, boric acid and EDTA;

[0028] And / or, the sample containing DNA is obtained by washing and lysing animal tissue, plant tissue or microbial culture, and the lysis buffer includes at least one of Tris-HCl, EDTA, sodium chloride, SDS and proteinase K.

[0029] The embodiment of the present invention discloses a device for purifying a sample containing DNA. Through electrophoresis, charged impurities such as proteins and polysaccharides migrate together with DNA, and the DNA is retained by a semipermeable membrane, while charged impurities with a smaller molecular weight can be removed through the semipermeable membrane, thereby achieving the purpose of removing charged impurities such as proteins and polysaccharides and purifying DNA. Since a gentle impurity removal method is adopted without violent vibration and other operations, the possibility of DNA breakage during the purification process is reduced, thereby achieving the purpose of maintaining the length of DNA while purifying DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the structure of a DNA purification device disclosed in an embodiment of the present invention;

[0031] Figure 2The electrophoresis gel images of DNA numbered 1 to 4 disclosed in the examples of the present invention;

[0032] Figure 3 The electrophoresis gel images of DNA No. 5 to 8 disclosed in the examples of the present invention;

[0033] Figure 4 The electrophoresis gel images of DNA numbered 10 to 11 disclosed in the examples of the present invention;

[0034] Figure 5 This is the DNA electrophoresis gel image No. 12 disclosed in the example of the present invention.

[0035] Figure 6 This is the electrophoresis gel image of DNA No. 13 to 15 disclosed in the examples of the present invention. DETAILED DESCRIPTION

[0036] The present invention provides an apparatus and method for purifying a sample containing DNA. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the purification. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0037] like Figure 1 As shown, the present application provides a device for purifying a sample containing DNA, comprising: a first liquid reservoir 1, a sample chamber 2, a second liquid reservoir 3 and an electrode assembly 4.

[0038] The first end 201 of the sample chamber 2 is placed in the first liquid reservoir 1, and the second end 202 of the sample chamber 2 is placed in the second liquid reservoir 3. The first end 201 of the sample chamber 2 has a first semipermeable membrane 21, and the second end 202 of the sample chamber 2 has a second semipermeable membrane 22. The sample chamber 2 forms an electrophoresis field between the first end 201 and the second end 202.

[0039] The first semipermeable membrane 21 and the second semipermeable membrane 22 are semipermeable membranes that are impermeable to the target separation substance from the inside to the outside of the sample chamber 2. In addition, the first semipermeable membrane 21 and the second semipermeable membrane 22 are both permeable to non-target separation substances.

[0040] Based on the scheme in the present application, the above-mentioned target separation is DNA, and the non-target separation includes but is not limited to charged impurities, cleaning fluid, etc. The charged impurities include proteins, polysaccharides, etc.

[0041] The first liquid reservoir 1 and the second liquid reservoir 3 are used to fill the electrophoresis buffer, and the two electrode ends of the electrode assembly 4 can be respectively placed in the first liquid reservoir 1 and the second liquid reservoir 3. When the first liquid reservoir 1 and the second liquid reservoir 3 are filled with the electrophoresis buffer, the electrophoresis buffer submerges the first end 201 and the second end 202 of the sample chamber 2.

[0042] In the working state, the electrode assembly 4 forms a closed-loop circuit and forms an electric field between the first end 201 and the second end 202 of the sample chamber 2. Under the action of the electric field, the charged substances in the sample in the sample chamber 2 migrate in the electric field, so that the non-target separation substances pass through the semipermeable membrane (including the first semipermeable membrane and the second semipermeable membrane), while the target separation substances cannot pass through the semipermeable membrane, thereby achieving the separation of the target separation substances and the purpose of purification.

[0043] The first semipermeable membrane 21 and the second semipermeable membrane 22 in the DNA purification device include, but are not limited to, ion exchange membranes, gel filtration membranes or electrophoresis membranes.

[0044] In some embodiments, DNA in the sample in the sample chamber 2 cannot pass through the first semipermeable membrane 21 and the second semipermeable membrane 22 , but biological molecules such as proteins and polysaccharides in the sample can pass through the first semipermeable membrane 21 and the second semipermeable membrane 22 .

[0045] In some embodiments, the electric field strength between the two electrodes of the electrode assembly 4 is 7V / cm-14V / cm, and the electrophoresis time maintained by the electrode assembly 4 is 30min-60min.

[0046] The electrode assembly 4 in some embodiments is an electrode assembly capable of realizing forward and reverse electrophoresis. When the electrophoresis condition is forward and reverse electrophoresis, the electrode assembly 4 can alternately generate forward current and reverse current. When the electrophoresis condition is forward and reverse electrophoresis, the electrode assembly maintains forward electrophoresis for a longer time than the reverse electrophoresis. The use of forward and reverse electrophoresis can improve the effect of DNA purification, and setting the time for maintaining forward electrophoresis to be longer than the time for maintaining reverse electrophoresis can facilitate the separation of DNA from proteins, polysaccharides, etc.

[0047] The DNA purification device in the present application can be placed in different ways according to different needs, including but not limited to horizontal placement and vertical placement.

[0048] In addition, the present application also discloses a method for purifying a sample containing DNA using the above-mentioned device.

[0049] Embodiment 1:

[0050] DNA purification experiments were performed using different electric field strengths and electrophoresis times. The electrophoresis instrument used was a Sage Science Pippin Pulse pulsed field electrophoresis instrument. The electrophoresis voltage was 42V, the electrophoresis time was 20 minutes, and the pulsed field electrophoresis program was set to perform forward and reverse electrophoresis. The specific settings of the pulsed field program are as follows:

[0051] Table 1:

[0052]

[0053] The electrophoresis time was set to 20 min.

[0054] In the table, V, A, B, C, D, E, F, and G respectively represent the following meanings:

[0055] V: electrophoresis voltage, unit is V.

[0056] A: Forward current time after electrophoresis starts, in msec.

[0057] B: Reverse current time after electrophoresis starts, in msec.

[0058] C: The incremental time added to A at each step, in msec.

[0059] D: The incremental time added to B at each step, in msec.

[0060] E: The incremental time added to C at each step, in msec.

[0061] F: The incremental time added to D at each step, in msec.

[0062] G: Number of steps in each cycle.

[0063] That is, after a forward electrophoresis of time A (i.e., 150msec), a reverse electrophoresis of time B (i.e., 50msec) is performed, and the first step is completed; next, in the second step, the forward electrophoresis time is A+C (i.e., 150+30=180msec); the reverse electrophoresis time is B+D (i.e., 50+10=60msec); in the third step, the forward electrophoresis time is A+C+E (i.e., 150+30+3=183msec); the reverse electrophoresis time is B+D+F (i.e., 50+10+1=61msec); and the above process is repeated until the total number of steps reaches 45, at which time a cycle is completed. Then repeat the above cycle until the set electrophoresis time is reached.

[0064] The electrophoresis voltage was set to 84 V, 42 V, 30 V, 18 V, and 0 V, and the electrophoresis time was set to 20 min, 40 min, and 60 min. Since the distance between the two electrodes was 6 cm, the electric field strength was 14 V / cm, 7 V / cm, 5 V / cm, 3 V / cm, and 0 V / cm when the voltage was 84 V, 42 V, 30 V, 18 V, and 0 V, respectively.

[0065] The specific steps are as follows:

[0066] S1. Preparation of DNA lysis buffer.

[0067] S11. Take 0.25 g of mouse muscle sample and grind it with liquid nitrogen until it becomes powder.

[0068] S12. Transfer the powder to a new 50 ml test tube and add 20 ml of lysis buffer (10 mM Tris-HCl, 25 mM EDTA and 100 mM NaCl) to the test tube and mix well.

[0069] S13. Add 1.5 ml of 20% SDS solution to the lysis buffer in the test tube and mix immediately.

[0070] S14. Add 400 μl of 20 mg / ml proteinase K solution and 60 μl of 10 mg / ml RNAse A solution to the test tube. Invert the test tube to mix well, and place the test tube at 50°C for 2 hours.

[0071] S15. After digestion is complete, the test tube is centrifuged at 10,000 g for 10 minutes.

[0072] S16. After centrifugation, take the supernatant DNA solution in the test tube and put it into a new 50ml tube. This DNA solution is used as the DNA solution to be purified.

[0073] S2. DNA separation and purification.

[0074] S201, taking a plurality of sample chambers 2, the operations of all the sample chambers 2 are the same, so as to form a plurality of experimental data and ensure the accuracy of the experimental data.

[0075] S202 , respectively open the first semipermeable membranes 21 of the DNA purification device, fill the sample chamber 2 with the DNA sample to be purified, the sample volume is 3 ml, and cover the first semipermeable membrane 21 .

[0076] S203, placing the sample chamber 2 containing the DNA sample between the first liquid reservoir 1 and the second liquid reservoir 3, and confirming that the contact parts between the tube wall of the sample chamber 2 and the first liquid reservoir 1 and the second liquid reservoir 3 are leak-proof.

[0077] S204 , add electrophoresis liquid 0.5X TBE (44.5 mM Tris (pH 7.6), 44.5 mM boric acid, 1 mM EDTA) into the first liquid reservoir 1 and the second liquid reservoir 3 , and make the electrophoresis liquid completely cover the first semipermeable membrane 21 and the second semipermeable membrane 22 .

[0078] S205, inserting electrodes into the first liquid reservoir 1 and the second liquid reservoir 3 respectively, the first liquid reservoir 1 corresponds to the negative electrode, and the second liquid reservoir 3 corresponds to the positive electrode.

[0079] S206, turning on the electrodes, and performing forward and reverse electrophoresis according to the set electrophoresis intensity and electrophoresis time.

[0080] This step is to separate impurities by forward and reverse electrophoresis and purify DNA.

[0081] S207, turn off the electrode.

[0082] S208, reverse the electrodes so that the first liquid reservoir 1 corresponds to the negative electrode and the second liquid reservoir 3 corresponds to the positive electrode.

[0083] S209, turn on the electrodes and perform direct current electrophoresis with an electric field strength of 14 V / cm and an electrophoresis time of 1 min.

[0084] This step is to further remove the DNA from the semipermeable membrane and reduce the adsorption of DNA on the membrane to achieve better results. This step is not a necessary step.

[0085] S210, take out the purification device, transfer all the liquid in the sample chamber 2 to a new 50 ml test tube to obtain a DNA sample.

[0086] S211. Select a DNA sample and in step S206, perform forward and reverse electrophoresis to purify the DNA at an electric field strength of 7 V / cm for 20 min, 40 min and 60 min; an electric field strength of 14 V / cm for 20 min, 40 min and 60 min; an electric field strength of 5 V / cm for 60 min; an electric field strength of 3 V / cm for 60 min; and an electric field strength of 0 V / cm for 0 min.

[0087] S212. Add 2 volumes of anhydrous ethanol to the DNA solutions in all 50 ml test tubes, turn the test tubes upside down, and shake them to precipitate the DNA.

[0088] S213. Transfer the DNA precipitate to new 2 ml test tubes, add 1 ml of 80% ethanol to each tube, invert the tubes, and shake them to wash.

[0089] S214, repeat the cleaning step.

[0090] S215. Transfer the DNA to new 2 ml test tubes and add an appropriate volume of EB solution (10 mM Tris-HCl) to the new 2 ml test tubes to dissolve the DNA.

[0091] S216. Use Qubit to measure the concentration.

[0092] S217. Use pulsed field electrophoresis to detect DNA length.

[0093] Table 2 below shows the DNA purification results using different electrophoresis fields and electrophoresis times:

[0094] Table 2:

[0095]

[0096] From the data results in Table 2 above, we can see that in terms of DNA OD value, the 260 / 280 of Experiment 8 is 1.86, and the 260 / 230 is 2.02, both within the normal range, and the DNA purity is good. However, the 260 / 230 of the extracted DNA No. 1-7 and No. 13-15 are all less than 2.0, and the purity is low.

[0097] Combination Figure 2 , Figure 3 and Figure 6 From the pulse field electrophoresis results shown in , it can be seen that the length of DNA obtained under the electric field conditions of 0 to 14 V / cm represented by numbers 1 to 8 and 13 to 15 is relatively long, ranging from 23 kb to 500 kb, or longer.

[0098] It should be noted that the 1-2 columns marked M1 and M2 on the left side of the gel image are distributed in a ladder-like manner. Each horizontal grid represents the corresponding DNA length. Generally, from top to bottom, the DNA length represented by each horizontal grid becomes smaller and smaller, thereby showing the distribution of DNA length. On the right is the DNA sample running on the gel, which is generally a diffuse band. The length of the sample DNA corresponds to the band of the DNA marker one by one. The length range of the sample DNA is indicated by the DNA marker. The longer the DNA length, the better the result.

[0099] Therefore, in summary, this embodiment shows that the conditions of Experiment 8 are more optimal. Under this condition, the purity of DNA is better and the length of DNA is longer. Therefore, the electric field strength of 7V / cm and the electrophoresis time of 60min are better electrophoresis conditions.

[0100] In addition, the electrode assembly in the method for purifying a sample containing DNA disclosed in the present application is an electrode assembly capable of realizing forward and reverse electrophoresis.

[0101] Embodiment 2:

[0102] Combined with the DNA purification experiment of plant specimens, it was verified that the recovery rate of forward and reverse electrophoresis was higher than that of unidirectional electrophoresis.

[0103] Plant samples were used for DNA purification experiments. The electric field strength was 14 V / cm and the electrophoresis time was 40 min. DNA was extracted by forward and reverse electrophoresis and unidirectional electrophoresis. The electrophoresis instrument used was the Pippin Pulse pulsed field electrophoresis instrument from Sage Science.

[0104] Since the distance between the two electrodes of this device is 6 cm, the electrophoresis voltage is 84 V when the electric field strength is 14 V / cm.

[0105] The forward and reverse electrophoresis was performed with an electrophoresis voltage of 84 V and an electrophoresis time of 40 min, and the pulse field electrophoresis program was set. The specific settings of the pulse field program are as follows:

[0106] Table 3:

[0107]

[0108] The electrophoresis time was set to 40 min.

[0109] For the meanings of V, A, B, C, D, E, F and G in the table, please refer to the explanation of Table 1.

[0110] The electrophoresis voltage was 84V, the electrophoresis time was 40min, and the one-way electrophoresis program was set to perform one-way electrophoresis. The specific settings of the electrophoresis program are as follows:

[0111] Table 4:

[0112]

[0113] The electrophoresis time was set to 40 min.

[0114] For the meanings of V, A, B, C, D, E, F and G in the table, please refer to the explanation of Table 1.

[0115] The specific steps are as follows:

[0116] S1. Isolation of cell nuclei.

[0117] Specifically, S11, take 1.5 g of plant leaf sample and put it into a pre-cooled 50 ml test tube, and place the 50 ml test tube in liquid nitrogen.

[0118] S12. Keep the 50 ml test tube in liquid nitrogen and use an iron spoon to crush the leaves in the test tube.

[0119] S13. Take out the 50 ml test tube from liquid nitrogen, add 15 ml of washing buffer (the washing buffer includes but is not limited to 10 mM Tris-HCl, 25 mM EDTA, 25 mM EGTA, 80 mM potassium chloride, 0.5 M sucrose, 0.5% (v / v) Triton X-100, 1% (w / v) PVP, 1 mM spermine, 1 mM spermidine and 0.2% (v / v) β-mercaptoethanol) to the 50 ml test tube in S12), and mix well.

[0120] S14. Use a homogenizer to break up the leaves in the test tube. The homogenizer can be Qiagen Tissue Ruptor.

[0121] S15. Add washing buffer to the crushed leaf solution to make up to 40 ml and mix well.

[0122] S16. Use a centrifuge with a swing rotor to centrifuge the solution from the previous step at a speed of 3500 rcf, a centrifugal time of 15 min, and a centrifugal temperature of 4°C.

[0123] S16. After centrifugation, discard the supernatant and add 1 ml of washing buffer to the remaining liquid to suspend the crude cell nuclear pellet, and add washing buffer to 40 ml.

[0124] S17. Use 100 μm and 40 μm filter sieves respectively to filter the solution obtained in the previous step to obtain a cell nucleus suspension.

[0125] S18. Use a centrifuge with a swing rotor to centrifuge the cell nucleus suspension obtained in the previous step. The centrifugal speed is 3500 rcf, the centrifugal time is 15 min, and the centrifugal temperature is 4°C. After the centrifugation is completed, the supernatant is discarded, and 1 ml of washing buffer is added to suspend the crude cell nucleus pellet. And the washing buffer is added to 40 ml.

[0126] S19. Repeat the previous step.

[0127] S20, centrifuge the solution in the previous step using a centrifuge with a swing rotor, discard the supernatant after centrifugation to obtain the cell nucleus precipitate. The centrifugal speed is 3500 rcf, the centrifugal time is 15 min, and the centrifugal temperature is 4°C.

[0128] S2, nuclear lysis.

[0129] S21. Add 20 ml of lysis buffer (10 mM Tris-HCl, 25 mM EDTA and 100 mM NaCl) to the cell nuclear pellet and mix well.

[0130] S22. Add 1.5 ml of 20% SDS solution to the lysis buffer and mix immediately.

[0131] S23. Add 400 μl of 20 mg / ml proteinase K solution and 60 μl of 10 mg / ml RNAse A solution to the solution obtained in the previous step. Invert the test tube to mix the solution, and place it at 50°C for 2 hours for digestion.

[0132] S24. After digestion, centrifuge the digested solution at 10,000 g for 10 min.

[0133] S25. After centrifugation, take the supernatant DNA solution into a new 50 ml test tube. This DNA solution is used as the DNA solution to be purified.

[0134] S3. DNA isolation and purification.

[0135] S301 , opening the first semipermeable membrane 21 of the DNA purification device, filling the sample chamber 2 of the device with the DNA sample to be purified, the sample volume is 3 ml, and covering the first semipermeable membrane 21 .

[0136] S302, placing the sample chamber 2 in the previous step between the first liquid reservoir 1 and the second liquid reservoir 3.

[0137] S303, add electrophoresis liquid 0.5X TBE (44.5 mM Tris (pH7.6), 44.5 mM boric acid, 1 mM EDTA) to the first liquid reservoir 1 and the second liquid reservoir 3, and make the electrophoresis liquid in the first liquid reservoir 1 submerge the first semipermeable membrane 21, and the electrophoresis liquid in the second liquid reservoir 3 submerge the second semipermeable membrane 22.

[0138] S304, inserting electrodes into the first liquid reservoir 1 and the second liquid reservoir 3 respectively, the first liquid reservoir 1 corresponds to the negative electrode, and the second liquid reservoir 3 corresponds to the positive electrode.

[0139] S305, turning on the electrodes, and performing forward and reverse electrophoresis, with an electrophoresis intensity of 14 V / cm and an electrophoresis time of 40 min.

[0140] This step is to separate impurities by forward and reverse electrophoresis and purify DNA.

[0141] S306, turn off the electrode.

[0142] S307, reverse the electrodes so that the first liquid reservoir 1 corresponds to the positive electrode and the second liquid reservoir 3 corresponds to the negative electrode.

[0143] S308, turn on the electrodes, set the electric field to 14 V / cm, and run the electrophoresis for 1 minute. The electrophoresis current is direct current.

[0144] This step further promotes the detachment of DNA from the semipermeable membrane, but it is not a necessary step.

[0145] S309, completely transfer the liquid in sample chamber 2 to a new 50 ml test tube to obtain the first purified DNA solution.

[0146] S310, the electrophoresis in S305 is changed to unidirectional electrophoresis, the electrophoresis intensity is 14 V / cm, and the electrophoresis time is 40 min. According to the above process, another portion of DNA electrophoresis purification is performed to obtain a second portion of purified DNA solution.

[0147] S311. Add 2 volumes of anhydrous ethanol to the first purified DNA solution and the second purified DNA solution respectively, and invert the test tubes and shake them to precipitate the DNA.

[0148] S312. Transfer the two groups of DNA precipitates to new 2 ml test tubes respectively, and add 1 ml of 80% ethanol to each of the two groups of 2 ml test tubes. Invert the test tubes and shake them to clean them.

[0149] S313. Repeat the previous step.

[0150] S314. Transfer the washed DNA solution to new 2 ml test tubes and add an appropriate volume of EB solution (10 mM Tris-HCl) to the test tubes to dissolve the DNA.

[0151] S315. Use Qubit to measure the concentration.

[0152] S316. Use pulsed field electrophoresis to detect DNA length.

[0153] Table 5 shows the two sets of DNA extraction results.

[0154] Table 5:

[0155]

[0156] It should be noted that the criteria for judging the advantages of DNA extraction using forward and reverse electrophoresis and unidirectional electrophoresis are mainly reflected in three aspects:

[0157] 1. High DNA purity.

[0158] It can be judged by DNA OD value or sequencing results. DNA OD value A260 / 280>1.8, and A260 / 230>2.0, indicating that the DNA purity is high.

[0159] Combined with this judgment standard, the experimental data in Table 5 show that the purity of DNA extracted by forward and reverse electrophoresis and one-way electrophoresis meets the requirements.

[0160] 2. The DNA is long.

[0161] Can be based on Figure 4 Judging from the DNA gel image shown in , the lengths of DNAs numbered 10 to 11 are all relatively long, ranging from 23kb to 500kb, or even longer.

[0162] 3. High DNA recovery rate.

[0163] For the same amount of DNA lysis buffer, the more total DNA obtained after purification, the higher the DNA recovery rate. Under the premise that the DNA length and purity meet the requirements, the higher the DNA recovery rate, the better the effect.

[0164] From the DNA recovery rate in Table 5, it can be seen that the DNA recovery rate of forward and reverse electrophoresis is twice that of unidirectional electrophoresis at the same electric field strength and electrophoresis time. Therefore, the forward and reverse electrophoresis method is conducive to improving the DNA recovery rate.

[0165] Generally speaking, the higher the DNA purity and the longer the length, the better the sequencing result; and the higher the DNA sequencing yield and the longer the N50, the better the result.

[0166] Comparative Example:

[0167] This comparative example verifies that the purity of DNA extracted by the DNA purification method of the present invention is equivalent to that of the phenol-chloroform extraction method.

[0168] Specifically, the phenol-chloroform DNA extraction method is used to extract DNA from animal tissues.

[0169] The steps of DNA extraction by phenol-chloroform extraction include:

[0170] S1001. Take 0.25 g of mouse muscle sample and grind it with liquid nitrogen until it becomes powder.

[0171] S1002. Transfer the powder to a new 50 ml test tube, add 20 ml of lysis buffer (10 mM Tris-HCl, 25 mM EDTA and 100 mM NaCl) to the test tube, and mix well.

[0172] S1003. Add 1.5 ml of 20% SDS solution to the lysis buffer and mix immediately.

[0173] S1004. Add 400 μl of 20 mg / ml proteinase K solution and 60 μl of 10 mg / ml RNAse A solution to the solution obtained in the previous step, invert the test tube to mix well, and place at 50°C for digestion for 2 hours.

[0174] S1004. After digestion is completed, centrifuge the digested solution at 10,000 g for 10 minutes.

[0175] S1005. After centrifugation, take the supernatant DNA solution into a new 50ml test tube.

[0176] S1006. Take 3 ml of the DNA solution from the previous step and put it into a new 15 ml test tube. Add 3 ml of phenol solution into the test tube. Place the test tube on a decolorizing shaker and shake at 15 rpm for 10 minutes.

[0177] S1007. After completion, place the centrifuge tube at room temperature and centrifuge at a centrifugal force of 4500 rcf for 10 minutes.

[0178] S1008. Use a wide-mouth pipette tip to transfer the supernatant solution to a new 15 ml test tube, and add 3 ml of phenol / chloroform / isoamyl alcohol solution (volume ratio of 25:24:1) to the test tube. Place the test tube on a decolorizing shaker and shake at 15 rpm for 10 minutes.

[0179] S1009. After completion, place the centrifuge tube at room temperature and centrifuge at a centrifugal force of 4500 rcf for 10 minutes.

[0180] S1010. Use a wide-mouth pipette tip to transfer the supernatant solution to a new 15 ml test tube, and add 3 ml of chloroform / isoamyl alcohol solution (volume ratio 24:1) to the test tube. Place the test tube on a decolorizing shaker and shake at 15 rpm for 10 minutes.

[0181] S1011. After completion, place the centrifuge tube at room temperature and centrifuge at a centrifugal force of 4500 rcf for 10 minutes.

[0182] S1012. Use a wide-mouth pipette tip to transfer the supernatant solution to a new 50 ml test tube, add 2 times the volume of anhydrous ethanol to the test tube, and gently invert to mix. A white flocculent precipitate will be produced. The white flocculent precipitate is the DNA precipitate.

[0183] S1012. Transfer the DNA precipitate to new 2 ml test tubes, add 1 ml of 80% ethanol to each test tube, invert the test tube, and shake the test tube for cleaning.

[0184] S1013. Repeat the previous step.

[0185] S1014. Transfer the DNA to new 2 ml test tubes and add an appropriate volume of EB solution (10 mM Tris-HCl) to dissolve the DNA.

[0186] S1015. Use Qubit to measure the concentration.

[0187] S1016. Use pulsed field electrophoresis to detect DNA length.

[0188] Table 6 shows the DNA extraction results using the phenol-chloroform extraction method.

[0189] Table 6:

[0190]

[0191] In Table 6, the 260 / 280 ratio of DNA No. 12 is 1.96, and the 260 / 230 ratio is 2.25. In Table 1, the 260 / 280 ratio of DNA No. 8 extracted using the scheme of the present invention is 1.86, and the 260 / 230 ratio is 2.02. The OD values ​​of the two DNAs are within the normal range, and the purity is good.

[0192] In summary, from the extraction results, it can be seen that the purity of DNA extracted by the DNA purification method of the present invention is equivalent to that extracted by the phenol-chloroform extraction method.

[0193] Sequencing effect verification

[0194] DNA No. 8 in Example 1 and DNA No. 12 in the comparative example were used to construct ONT libraries and sequenced on a sequencing machine to further compare the purification effects of the two methods.

[0195] Use nanopore LSK114 library construction kit to build the library. For the steps, see Ligationsequencing gDNA (SQK-LSK114).

[0196] The sequencing results are as follows:

[0197]

[0198] It can be seen from the sequencing results that under the same sequencing conditions, although the total data volume of sample No. 8 is slightly lower than that of sample No. 12, the N50 measured by sample No. 8 is longer, 1.38 times that of sample No. 12, and the yield of long-fragment DNA (>50kb>100kb>150kb) is also higher. The data volume of >150kb of sample No. 8 is 2.4 times that of sample No. 12. Therefore, compared with the conventional phenol-chloroform extraction method, the DNA purification method of the present invention has significant advantages in extracting and sequencing long-fragment DNA.

[0199] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0200] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A device for purifying a sample containing DNA, characterized in that: include: A sample chamber, wherein the sample chamber is used to hold a sample containing DNA to be processed, a first semipermeable membrane is provided at a first end of the sample chamber, a second semipermeable membrane is provided at a second end of the sample chamber, and both the first semipermeable membrane and the second semipermeable membrane are membrane structures, and non-target separated substances can pass through the direction from the inside to the outside of the sample chamber, but target separated substances cannot pass through, an electrophoresis field is formed between the first end and the second end of the sample chamber, the target separated substance is DNA, and the non-target separated substances include charged impurities; A first liquid reservoir and a second liquid reservoir, wherein the first end of the sample chamber is sealed in the first liquid reservoir, the second end of the sample chamber is sealed in the second liquid reservoir, and the first liquid reservoir and the second liquid reservoir are used to contain electrophoresis buffer solution; An electrode assembly, wherein one electrode of the electrode assembly extends into the first liquid reservoir, and the other electrode of the electrode assembly extends into the second liquid reservoir to form a loop for electrophoresis of a sample containing DNA.

2. The device for purifying a sample containing DNA according to claim 1, characterized in that: The electric field strength between the two electrodes of the electrode assembly is 3V / cm-14V / cm, and the electrophoresis time maintained by the electrode assembly is 30min-60min.

3. The device for purifying a sample containing DNA according to claim 2, characterized in that: The electric field strength between the two electrodes of the electrode assembly is 7 V / cm, and the electrophoresis time maintained by the electrode assembly is 60 min.

4. The device for purifying a sample containing DNA according to claim 1, characterized in that: The electrode assembly is an electrode assembly that can alternately generate forward current and reverse current.

5. The device for purifying a sample containing DNA according to claim 4, characterized in that: When the electrophoresis condition is forward and reverse electrophoresis, the electrode assembly maintains forward electrophoresis for a longer time than the reverse electrophoresis.

6. A method for purifying a sample containing DNA, using the device according to any one of claims 1 to 5, characterized in that: include: placing a sample containing DNA in the sample chamber, and adding electrophoresis buffer to the first reservoir and the second reservoir; Applying an electric field between two electrodes of the electrode assembly to perform forward and reverse electrophoresis; The liquid in the sample chamber after electrophoresis is further purified to obtain DNA.

7. The method for purifying a sample containing DNA according to claim 6, characterized in that: The duration of the forward electrophoresis of the forward and reverse electrophoresis is longer than that of the reverse electrophoresis.

8. The method for purifying a sample containing DNA according to claim 7, characterized in that: The forward electrophoresis and the reverse electrophoresis of the forward and reverse electrophoresis are performed alternately at least twice, and within a single cycle, the total duration of the forward electrophoresis is greater than the total duration of the reverse electrophoresis.

9. The method for purifying a sample containing DNA according to claim 6, characterized in that: The step of further purifying the liquid in the sample chamber after electrophoresis comprises: using ethanol or ethanol aqueous solution to precipitate the DNA, wherein the volume ratio of the ethanol or ethanol aqueous solution to the liquid in the sample chamber is (1-3): 1, and the volume fraction of ethanol in the ethanol aqueous solution is not less than 90%; And / or, the step of further purifying the liquid in the sample chamber after electrophoresis further includes: washing the DNA precipitate with an ethanol aqueous solution, the number of washings is 1 to 3 times, and the volume fraction of ethanol in the ethanol aqueous solution is not less than 75%; And / or, the cleaning further includes a dissolving step, wherein the dissolving solution is water, EB solution, Tris-HCl solution or TE buffer; And / or, the electrophoresis buffer uses at least one of Tris, boric acid and EDTA; And / or, the sample containing DNA is obtained by washing and lysing animal tissue, plant tissue or microbial culture, and the lysis buffer includes at least one of Tris-HCl, EDTA, sodium chloride, SDS and proteinase K.

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